Novel chitinase-producing bacteria and plants

ABSTRACT

Novel bacteria strains are decribed which are created by the introduction of DNA encoding for the production of chitinase, an enzyme capable of degrading chitin present in fungi and nematodes. The strains have utility in producing chitinase for the purpose of inhibiting plant pathogens. Novel pathogen-resistant plants are also described which are created by introduction of DNA encoding for the production of chitinase.

This invention was made with Government support under Grant No.ISI-8560311 awarded by the National Science Foundation. The Governmenthas certain rights in this invention.

This application is a continuation-in-part of pending U.S. Ser. No.593,691 in the names of Trevor V. Suslow and Jonathan D. G. Jones, filedMar. 26, 1984, for NOVEL CHITINASE-PRODUCING BACTERIA, now U.S. Pat. No.4,751,081.

This invention relates to novel man-made bacterial strains which producechitinase, an enzyme which degrades chitin. This invention furtherrelates to the use of such strains as a means to inhibit soil fungi andnematodes and to enhance plant growth by biological control of plantpathogens. This invention additionally relates to the introduction ofchitinase activity into plants and to plants which have been renderedresistant to plant pathogens as a result of such introduction.

The soil contains a wide variety of life forms which can interact withplants, including bacteria, fungi and nematodes. These life forms areespecially abundant in the rhizosphere, the area of the soil thatsurrounds and is influenced by the plant roots. As used herein the termrhizosphere embraces the rhizoplane, the root-soil interface includingthe surface of the root. The term rhizobacteria, as used herein, refersto bacteria adapted to the rhizosphere. The interactions between thesesoil inhabiting life forms are complex, some being antagonistic andothers being mutually beneficial.

The interactions between plants and the various soil life forms aresimilarly complex, in some instances helpful to the plant and in otherinstances deleterious to the plant. Fungi harmful to plants (fungalpathogens) include fungal species from a wide variety of genera,including Fusarium, Pythium, Phytophthora, Verticillium, Rhizoctonia,Macrophomina, Thielaviopsis, Sclerotinia and numerous others. Plantdiseases caused by fungi include pre- and post-emergence seedlingdamping-off, hypocotyl rots, root rots, crown rots, vascular wilts and avariety of other forms of symptom development. Nematodes harmful toplants (nematode pathogens) include nematode species from the generaMeloidogyne, Heterodera, Ditylenchus, Pratylenchus. Plant diseasescaused by nematodes include root galls, root rot, lesions, "stubby"root, stunting, and various other rots and wilts associated withincreased infection by pathogenic fungi. Some nematodes (e.g.Trichodorus, Lonoidorus, Xiphenema) can serve as vectors for virusdiseases in a number of plants including Prunus, grape, tobacco andtomato.

Various approaches are available for attempting to control deleteriousfungi and nematodes. One method, long known in the art, is chemicaltreatment of soil or plants with fungicides or nematicides. Anothermethod is application of certain naturally occurring bacteria whichinhibit or interfere with fungi or nematodes. See, in general, K. F.Baker and R. J. Cook, Biological Control of Plant Pathogens, Freeman andCo. (1974) for a description of fungi and nematodes and theirinteraction with plants, as well as a description of means forbiological control of fungal and nematode pathogens.

One approach to biocontrol of fungal and nematode pathogens is based onthe widespread presence of chitin as an integral part of the cell wallsof fungi and the outer covering of nematodes or nematode eggs ornematode cysts. Chitin is an unbranched polysaccharide polymerconsisting of N-acetyl-D-glucosamine units. It is insoluble in water,dilute mineral acids and bases but can be broken down enzymatically bychitinase, the degradation products being soluble monomers or multimersof N-acetyl-D-glucosamine. Chitinase is produced by certain naturallyoccurring bacteria and fungi and there have been reports of the role ofchitinase in the suppression of pathogens.

R. Mitchell and M. Alexander, "The Mycolytic Phenomenon and BiologicalControl of Fusarium in Soil", Nature, 190, 109-110 (1961) describesnaturally occurring mycolytic, or fungi-lysing, soil bacteria (generaBacillus and Pseudomonas) which suppress soil Fusarium by means ofchitinase activity.

B. Sneh, Use of Rhizosphere Chitinolytic Bacteria for BiologicalControl", Phytopath. Z., 100, 251-56 (1981) discloses naturallyoccurring chitinolytic isolates identified as Arthrobacter sp. andSerratia liquifaciens. Sneh also discloses introduction of achitinolytic bacterial strain from the genus Arthrobacter into therhizosphere to protect carnation seedlings from Fusarium wilt.

A. H. Michael and P. E. Nelson, "Antagonistic effect of soil bacteria onFusarium roseum culmorum", Phytopathology, 62, 1052-1056 (1972)discloses similar control with a naturally occurring Pseudomonasspecies.

J. Monreal and E. T. Reese, "The Chitinase of Serratia marcescens",Canadian Journal of Microbiology, 15, 689-696 (1969) describes aSerratia marcescens bacterial strain (QMB1466) selected as the mostactive chitinase producer out of a number of naturally occurringbacterial and fungal strains tested. Other strains tested whichdisplayed some chitinase activity included bacterial strains from thegenera Enterobacter and Streptomyces, and fungal strains from the generaAspergillus, Pencillium and Trichoderma. Chitinase is characterized asan induced enzyme system in strain QMB1466, i.e. the yields of chitinaseproduced by the strain were higher when chitin was present. Monreal etal. reports at p. 692 that chitinase production on a chitin medium isrepressed by the addition of other carbon-containing metabolites, e.g.sugars, to the medium. The Serratia marcescens enzyme system isdescribed as extracellular and including endochitinase, a chitobiase anda "factor" for hydrolysis of "crystalline" chitin.

The naturally occurring Serratia marcescens chitinase system is furtherdescribed in R. L. Roberts and E. Cabib, "Serratia Marcescens Chitinase:One-Step Purification and Use for the Determination of Chitin",Analytical Biochemistry, 127, 402-412 (1982).

J. D. Reid and D. M. Ogrydziak, "Chitinase- Overproducing Mutant ofSerratia marcescens", Applied and Environmental Microbiology, 41,664-669 (1981) describes work with a mutant of Serratia marcescens,strain IMR-1E1, obtained by mutation of strain QMB1466. The mutant hadincreased chitinase activity compared to strain QMB1466, as measured byzones of clearing on chitin-agar plates. 0n page 664 Reid et al. refersto the "high rate of reversion of IMR-1E1 to decreased levels ofchitinase production."

C. I. Kado and P. F. Lurquin, "Prospectus for Genetic Engineering inAgriculture", Phytopathogenic Prokaryotes, Vol. 2, M. S. Mount and G. H.Lacy eds., 309 (1982), while not discussing the role of chitinase incontrolling chitin-containing pathogens, notes the possibility of adifferent approach to controlling fungi, namely, inserting into bacteriagenes coding for compounds which inhibit chitin synthase in fungi. Thatis, the compound chitin synthase, necessary for production of chitin infungi, would be inhibited by the bacterial compounds.

P. M. Miller and D. C. Sands, "Effects of Hydrolytic Enzymes onPlant-parasitic Nematodes", Journal of Nematology, 9, 192-197 (1977)describes the effect of chitinase, obtained from a commercial supplier,on certain nematodes. Miller et al. discloses that chitinase hydrolyticenzymes are toxic to certain nematodes, in particular Tylenchorhynchusdubius, the toxicity being greater in aqueous solution than in soil.

There are a number of limiting factors and disadvantages with respect towork to date on biological control of plant pathogens usingchitinase-producing bacteria introduced into the soil. First is theinability to regulate the production of chitinase in the introducedbacteria in such a way that proper amounts of chitinase are produced.Second is the limited ability of many of such bacteria to colonize andpersist in the rhizosphere of host plants, a key consideration foreffective biocontrol. Particularly important in this respect is theability of biocontrol bacteria to colonize the roots of host plantseffectively, the roots being the site of much plant-pathogeninteraction. Third is that chitinase production is repressed in thepresence of other carbon sources, e.g. metabolites released by the root.Another problem, at least as to mutants, is reversion to formsexhibiting decreased levels of chitinase production.

There have been a number of reports of methods for introducing foreignDNA into plants. One approach is introduction by transformation usingAorobacterium, in particular Agrobacterium tumefaciens; M. Bevan et al.,Ann. Rev. Genet., 16, 357-384 (1982); L. Ream et al., Science, 218,854-859 (1982). This introduction may be carried out by cocultivation ofplant protoplasts with Agrobacterium, followed by plant regeneration;Marton et al., Nature, 277, 129-131 (1979); R. B. Horsch et al.,Science, 223, 496-498 (1984). The introduction may also be carried outusing binary vectors; A. Hoekema et al., Nature, 303, 179-180 (1983); P.van den Elzen et al., Plant Mol. Biol., 5, 149-154 (1985); M. Bevan,Nucl. Acids Res., 12, 8711-8721 (1984). Where the foreign DNA is from anon-plant source, the foreign DNA (structural gene, i.e., encodingsequence) may be fused to a plant promoter; L. Herrera-Estrella et al.,Nature, 303, 209-213 (1983); R. T. Fraley et al., Proc. Natl. Acad.Sci., 80, 4803-4807 (1983); J. T. Odell et al., Nature, 313, 810-812(1985); J. Jones et al., EMBO J., 4, 2411-2418 (1985).

The present invention comprises novel man-made bacteria, in particularrhizobacteria, which have the ability to produce chitinase as the resultof introduction into the bacteria of foreign DNA encoding for chitinaseactivity. The foreign DNA is isolated from a foreign source, bacterialor otherwise, or is substantially homologous to such DNA. The novelbacteria can be prepared using various means, including the use ofappropriate vectors, for introducing the foreign DNA, and thus thecapacity to produce chitinase, into a bacterial cell or a parent of abacterial cell, under conditions where chitinase activity is expressed.The vectors are used to clone and introduce the foreign DNA whichencodes for chitinase activity. The novel bacteria can be used tointroduce chitinase into the soil, particularly into the soilrhizosphere, thereby providing a means of inhibiting chitin-containingor chitinase-sensitive plant pathogens, including fungi and nematodes,and thereby also providing a means of enhancing the growth and wellbeing of plants sensitive to such pathogens.

The present invention provides a means of overcoming limitations of theprior art methods of bacterial control of chitinase-sensitive plantpathogens. The invention provides a means of introducing sufficientchitinase production capacity into a strain. The invention also providesa means to introduce chitinase capacity into strains best suited tofunction in the soil and rhizosphere, in particular root colonizingrhizobacteria strains. Further, in accordance with the invention, theproblem of reversion of modified strains to wild type is overcome inthat the novel strains of the invention result from the actualintroduction of genetic material, rather than from mutation.Additionally, the invention provides the means to overcome the problemof repression of chitinase activity in the presence of root exudates orother carbon sources, in that regulatory systems can be employed whichrender the bacterial cell insensitive to such repression.

The present invention further comprises a method of introducing intoplants the ability to produce chitinase as the result of introductioninto the plant of foreign DNA encoding for chitinase activity.Accordingly, the invention also comprises a method of inhibitingchitinous (chitinase-containing) plant pathogens by such introduction offoreign DNA under conditions whereby the plant produces (expresses)chitinase in active form. More particularly, plants may be protectedagainst fungi, nematodes, insects and disease agents. As used herein,"in active form" means chemically active (capable of degrading chitin)or biologically active (capable of inhibiting chitinous plantpathogens).

The foreign DNA to be introduced into the plant is isolated from aforeign source, in particular a bacterial source, or is substantiallyhomologous to such DNA. The foreign DNA is introduced into the plantusing plant transformation techniques, in particular using Agrobacteriumprocedures whereby the foreign chitinase DNA is first introduced intoAgrobacterium in an appropriate vector, in accordance with the method ofthe invention, and then introduced into the plant by transformation withAgrobacterium. Such introduction into the plant is preferably carriedout where the foreign DNA is fused to a plant promoter such that theforeign DNA (the structural gene) is under the transcriptional controlof the plant promoter. Preferred sources of foreign chitinase DNA forplant transformation are ATCC #39637 and ATCC #67152.

The present invention in addition comprises plants which have beenrendered resistant to, or capable of inhibiting, chitinous plantpathogens by virtue of introduction of foreign chitinase-encoding DNAinto the plant under conditions whereby the transformed plant expresseschitinase in active form. The invention also comprises progeny of suchtransformed plants. As used herein "transformed plant" means a plantinto which foreign DNA has been introduced. Any transformable plant maybe transformed in accordance with the invention. The term "plant" asused herein includes whole plants, plant parts, seeds, plant cells,plant calli and plant tissue cultures. Preferred plants fortransformation in accordance with the invention include tobacco,Brassica spp., soybean, sugarbeet, cotton, tomato, pepper, alfalfa,potato, wheat, barley, rice and corn.

The novel bacterial cells of the invention are made by introduction offoreign DNA, or heterologous DNA, which codes for production of, orexpression of, the enzyme chitinase. The term "chitinase" is used hereinto mean chitin-degrading enzyme, the term "chitin-degrading" embracingboth chitin-hydrolyzing and chitin-solubilizing. The term chitinase DNA"is used herein to mean DNA which encodes for chitinase, and embracesforeign chitinase DNA obtained directly or indirectly from a sourceorganism, including bacteria, fungi and plants, as well as DNA whichregardless of source is substantially homologous to such foreignchitinase DNA. "Chitinase activity", or "chitinolytic activity", as usedherein, means the ability or capacity of a bacterial cell to producechitinase. Such chitinase can be secreted by the bacteria into theimmediate environment.

Chitinase DNA can be obtained from a wide variety of naturally occurringbacteria which are known to or can be shown to produce chitinase,including bacteria from the genera Serratia, Bacillus, Pseudomonas,Arthrobacter, Enterobacter, and Streotomyces. Bacterial strainscontaining chitinase DNA have been known and available from laboratoriesor collections for years. For instance, chitinase-producing Serratiamarcescens strain QMB1466, which was described by Monreal et al. in 1969and by Reid et al. in 1981, (in each case the reported source of thestrain being the U.S. Army Natick Laboratory Culture Collection) isavailable from a number of sources, including the American Type CultureCollection at Rockville, Maryland (ATCC 990). Chitinase-containingbacterial strains are also readily obtainable by known techniques byvirtue of their widespread distribution in nature. Such strains ingeneral are found in soil, on plants, on insects and in water systems,as well as in other places where chitin is present. For example,chitinolytic bacteria can be isolated from the rhizosphere of a widevariety of plants including sugar beet, cotton, bean or carnation.Chitinase-producing bacteria can also be obtained from root surfaces,fungal resting structures (e.g. sclerotia, chlamydospores), nematode eggmasses, insect or arthropod exo-skeleton and irrigation water.

Isolation of bacterial strains containing chitinase DNA can beaccomplished by a number of techniques, including direct isolation onchitin-containing media, enrichment or baiting with chitin or fungalhyphae. These techniques are common and known to those skilled in theart. Chitinase-producing fungi can be isolated from sources such asthose stated above for chitinolytic bacteria, again using standardtechniques for plating fungi. See, in general, J. Tuite, PlantPathological Methods: Fungi and Bacteria, Burgess Publishing Co. (1969)with respect to techniques for isolation of bacteria and fungi.

Foreign chitinase DNA for conferring chitinase activity on a host, orrecipient, bacterium can be obtained directly from a source organism,e.g. bacteria, fungi, yeast, insect or plant, using techniques of genomefragmentation and DNA isolation known to those skilled in the art. Forinstance, for a bacterial source organism, isolated as explained above,total bacterial DNA (that is, the entire genome including chromosomalDNA and extra-chromosomal DNA) is isolated by standard techniques, e.g.lysis of bacteria in the presence of appropriate detergents, proteasesand chelating agents, followed by phenol and chloroform extractions andprecipitation with ethanol. The isolated DNA is partially digested tovarious degrees with an appropriate restriction enzyme or enzymesselected on the basis of appropriate sites in the cloning vector whichis to be used. The products of the digestion process are fractionated bystandard techniques, for instance on a glycerol gradient. Fractionscontaining DNA in an appropriate size range, e.g. about 22 to about 32kb (kilo bases), are selected for insertion into an appropriate vectorusing known techniques, for instance as described below, thus yielding agenomic library (consisting of cosmid clones, in the case of a cosmidvector).

An alternative to obtaining chitinase DNA directly by genomefragmentation of a source organism is obtaining chitinase DNA indirectlyby isolating, from the source organism, messenger RNA (mRNA)corresponding to chitinase DNA. A cDNA (copy DNA) library can beprepared from the mRNA, using reverse transcriptase in accordance withtechniques known to those skilled in the art, and inserted into anappropriate cDNA expression vector, such that clones encoding chitinaseactivity could be detected by clearing of chitin on plates.

The choice of particular vector turns on a number of considerationsknown to those skilled in the art, including the size of the fragment,nature of the host, number and position of restriction sites desired,and selection marker or markers desired. Techniques for introduction ofDNA into a vector and subsequent introduction of the vector into thehost bacteria are known to those skilled in the art. See in general T.Maniatis et al, "Molecular Cloning, a Laboratory Manual," Cold SpringHarbor Publications, 1982 (hereinafter Maniatis) with respect totechniques for insertion of DNA fragments into a host bacterium (as wellas with respect to general techniques for fragmentation andfractionation of a genome.)

Introduction of foreign DNA into the host bacteria results in thecreation of a bank of modified, i.e. transformed or transduced, hostbacteria which can be screened for chitinase DNA. In many cases the hostbacteria will be E. coli. The screening can be carried out by platingthe host strains on a medium which contains chitin, e.g. colloidalchitin. The development of zones of clearing in the chitin around acolony is evidence that the colony is chitinolytic. Microscopicexamination showing dissolution of surrounding chitin particles isfurther evidence. Alternative means to screen will be apparent to thoseskilled, e.g. plating on a fungal lawn, or chemical tests to show thepresence of chitinase.

For those bacteria shown by screening to exhibit chitinase activity,there can be optionally employed a subsequent subcloning to reduce thequantity of cloned DNA which is not involved in the coding forchitinase. An appropriate enzyme digestion is carried out and thedigestion products ligated to another more convenient cloning vector,e.g. one with high copy number, and the ligation products are againtransformed into E. coli bacteria by known techniques. Transformants arescreened for chitinase production as described above.

After the cloning and any subcloning, if desired, the chitinase DNA canbe transferred from the first host (transferor or donor) bacterial cellinto a second host (transferee or recipient) bacterial cell. Thistransfer can be accomplished using known techniques, for instance byconjugation using helper plasmids to mobilize the plasmid into thetransconjugant cell, the specifics depending on the transferorbacterium, the recipient bacterium, and the cloning vector which is usedto propagate the chitinase DNA. For instance, if the chitinase DNA iscloned on an IncP (incompatibility group P) type plasmid derivative,such as pLAFRl, transfer to a second host strain in many instances canbe accomplished by conjugation, e.g. using a helper plasmid such aspRK2013. See in general G. Ditta et al, "Broad Host Range DNA cloningsystem for Gram-negative bacteria: Construction of a gene bank ofRhizobium meliloti", Proc. Natl. Acad. Sci., 77, 7347-7351 (1980)(hereinafter Ditta), with respect to conjugation using helper plasmids.Where the intended use of the bacteria modified to have chitinaseability is in control of plant pathogens residing in the soil, thebacteria of choice will normally be rhizobacteria. In that eventchitinase DNA is transferred from the first host, normally E. coli, intothe second host rhizobacterial strain.

Depending on the systems and circumstances involved in transferring avector containing chitinase DNA from one bacterial cell to another,various techniques known to those skilled in the art may be used toensure proper expression of the chitinase DNA in the host. For instance,an effective regulatory or promoter system will be necessary to bringabout proper expression, that is, to ensure that the production ofchitinase, encoded for by foreign chitinase DNA, can be brought aboutunder conditions where chitinase production is desired. If the promoterfrom the source organism (i.e, the promoter which normally works in thesource organism with the foreign chitinase DNA) is not effective in thehost, it may be necessary to incorporate into the vector a regulatorysystem different from that which controlled the foreign DNA in thesource organism. A promoter system of choice may be one which allows thebacterial cell to produce chitinase in a manner insensitive to thepresence of carbon sources, e.g. root metabolites, in the immediateenvironment. That is, the cell can be made to produce chitinaseconstitutively. Various other techniques to enhance chitinase activityin the modified cell may be employed as well, e.g. multicopy vectors ormeans to enhance secretion of the chitinase from the cell.

Plasmids containing chitinase DNA, i.e. clones or chimaeric plasmids,can be introduced into a bacterial host by transformation, e.g. usingCaCl₂, the transformed cell being called the transformant. The plasmidmay be a cosmid vector containing chitinase DNA, i.e. a cosmid clone,and if so it can also be introduced into the bacterial cell bytransduction, the product cell being the transductant.

The particular adaption of rhizobacterial cells to the rhizosphere isrelated to their ability to multiply and compete at the root-soilinterface or the root surface, or in the intercortical cell spaces. Rootcolonizing rhizobacteria typically reach population densities of 10⁴ orgreater colony forming units (cfu) per mg of root tissue, from lowinitial populations, during the first several weeks of plant growth.Various rhizobacteria have been described, including strains from thegenera Pseudomonas (in particular P. fluorescens and P. putida),Agrobacterium, Enterobacter and Alcaligenes. See in general T. Suslow,"Role of Root-Colonizing Bacteria in Plant Growth", PhytopathogenicProkaryotes, Vol. 1, M. S. Mount and G. H. Lacy eds., 187-223 (1982) fora discussion of root colonizing rhizosphere bacteria and theirproperties. The choice of root colonizing strain to receive chitinaseDNA will turn on the plant to be protected, the pathogen or pathogens tobe protected against, the method of application, and the culturalpractices related to the crop of interest.

For bacterial strains which already have some chitinase activity,introduction of chitinase DNA in accordance with the present inventionserves to enhance chitinase activity in the host. Other bacteria alreadyhave anti-fungal (fungicidal) or anti-nematodal (nematicidal) capacityby some mechanism other than chitinase activity, in which caseintroduction of chitinase DNA confers chitinase activity and enhancesanti-pathogen ability.

The present invention can also be used in combination with theintroduction of some other foreign DNA, that is foreign DNA other thanchitinase DNA, into a bacteria or plant. For instance, in the case ofrhizobacteria, such other foreign DNA could provide the host with someother form of anti-pathogen activity or with some other means to allowit to enhance the soil environment to the benefit of the plant.

The present invention is of agricultural use as a means for theproduction of chitinase, including the production of chitinase as anantibiotic for the purpose of degrading or otherwise inhibiting,repelling or killing plant pathogens harmful to a wide variety ofagricultural crops. The invention has particular utility for inhibitingchitinase-sensitive fungi or nematodes (that is, fungi or nematodeswhich are inhibited, repelled or destroyed in the presence ofchitinase), where such fungi or nematodes or their activities in soil oron plant surfaces are harmful to plants. Regardless of the mechanism bywhich such pathogens are injurious to plants, their inhibition serves toenhance plant growth and health.

Bacteria, and particularly rhizobacteria, modified in accordance withthe present invention and grown to sufficient proportions, e.g., byfermentation, can be used to combat chitin-containing soil pathogens byapplication of the bacteria to soil, seeds, vegetative plant parts orirrigation water. For example, mycolytic bacteria created in accordancewith the invention can be used in such ways to attack or inhibit fungi.The modified bacteria can be applied in various formulations containingagronomically acceptable adjuvants or carriers in dosages andconcentrations chosen to maximize the beneficial effect of therhizobacteria.

For application to soil, to soil mixes, or to artificial plant growthmedia, the modified bacteria may be applied as a powder or granule in asuitable carrier. Alternatively, the modified bacteria may be applied asa suspension or dispersion, e.g. as an aqueous suspension with asuitable protectant such as methycellulose, dextran, dextrin, alginate,magnesium silicate. The modified bacteria may also be applied as awettable powder.

For application to seeds, the modified bacteria may be applied as partof a seed coating composition, for instance mixed with xanthan gum,magnesium silicate, methylcellulose, gum arabic, polyvinyl pyrollidone,dextrins or dextrans. In addition, small amounts of partially hydrolyzedchitin may be added to the pelleting mix, dust granule, suspension, orwettable powder to enhance chitinase production. See in general T.Suslow et al., "Rhizobacteria of sugar beets: effects of seedapplication and root colonization on yield", Phytopathology, 72, 199-206(1982); and, J. Kloepper et al., "Development of a powder formulationfor inoculation of potato seed pieces", Phytopathology, 71, 590-592(1981), for a discussion of rhizobacteria and seed coating compositions.

Bacteria into which chitinase capability has been introduced by thisinvention may also be applied to the above-ground surface of a plant,e.g., the leaf or stem surface, either to permit the modified bacteriato travel or spread to the roots or to inhibit chitinase-sensitivepathogens which may be present on blossoms or plant surfaces, forinstance, fungal pathogens such as Botrytis, Monilinia, Alternaria, andCercosoora. Blossoms of Prunus sp., in particular, provide an idealenvironment for the growth of epiphytic bacteria, e.g. Pseudomonassyringae or Erwinia herbicola, that have the ability to produceinhibitory levels of chitinase. Similar results can be obtained byintroducing chitinase-producing capacity directly into the plant.

The method of the invention can also be used for introduction ofchitinase genes into species of Rhizobium which enter into anitrogen-fixing symbiosis within the nodules of leguminous plants. Thenodules are frequently the point of entry of pathogenic fungi andnematodes.

The method of the invention additionally provides a means to introducechitinase DNA into a bacteria, e.g. Aorobacterium, which is used totransfer the foreign DNA to plants. Other known means of transformingplants are also available to transfer chitinase DNA to plants and theinvention is not limited to any given method of transformation. Suchtransfer results in a direct means for the plant to inhibitchitinase-sensitive plant pathogens, either alone or in conjunction withbacteria modified to have chitinase ability. A particularly attractiveform of such transfer is one where the chitinase DNA is expressed by theplant only at the site of pathogen attack, e.g. only in the root cells.

Both of the above applications (introduction of chitinase activity intoRhizobium or plants) would involve subcloning the chitinase genes andbringing them under the control of different regulatory sequences fromthose which act in the source organism. For example, elevated expressionin E. coli could be brought about by using the lac Z system(B-galactosidase structural gene promoter). In nodules elicited byRhizobium a nitrogenase promoter could be used, and in plant leaves thepromoter of a highly expressed leaf gene could be used. Any of a numberof such plant promoters can be used in accordance with the invention. Asused herein, the term "plant promoter" means a promoter, whether ofplant source or otherwise, which can function in a plant, i.e., whichcan control or regulate the transcription of a structural gene within aplant cell. As used herein, the term "promoter" means a DNA sequencewhich can control the level of transcription of a downstream DNAsequence which corresponds to a structural gene (encoding region). Plantpromoters may be fused to structural genes by cutting and ligating ofDNA sequences, together with substitution of specific nucleotide(s) asneeded in ways known in the art. The following plant promoters,preferred for use in regulating chitinase DNA in plants, have been fusedto chitinase DNA and introduced into plants resulting in chitinaseproduction by the plants, in accordance with the invention: nopalinesynthase promoter (from Agrobacterium tumefaciens); chlorophyll a/bbinding protein promoter; ribulose bisphosphate carboxylase smallsubunit promoter; and cauliflower mosaic virus 35S promoter.

Plants transformed with chitinase DNA may be characterized or subjectedto assays in various ways. The presence of messenger RNA correspondingto chitinase DNA may be assayed in known ways (e.g., Northernhybridization, primer extension assay) as a measure of transcription ofthe introduced DNA. The presence of chitinase protein may be assayed inknown ways (e.g., SDS-PAGE) as a measure of translation. Chitinaseproduced by the plant may be assayed for chemical activity by measuringthe capacity of chitinase produced by the plant to hydrolyze chitin. Thebiological activity of the chitinase may be assayed by various bioassayswhich can be used to determine the effect of chitinase produced by theplant on chitinous plant pathogens. One such bioassay is described inExample 4(f).

EXAMPLES 1. Introduction of Chitinase DNA into E. Coli

The overall procedure was to construct a set of random cosmid clonesfrom the Serratia marcescens genome which would cover the entire genomeseveral times over in such a way that statistically there was at least a99% chance of covering every DNA sequence in the genome. Clones carryingan entire chitinase gene were inserted in E. coli, which is quiteclosely related, taxonomically, to S. marcescens. The work involved inisolating clones which carry chitinase DNA had the following steps, asexplained in detail below.

(a) Isolating total S. marcescens DNA.

(b) Partial digesting of S. marcescens DNA.

(c) Purifying a fraction of the partial DNA digest in which the DNAfragment size was 22 kb - 32 kb.

(d) Ligating the purified DNA to a cosmid cloning vector.

(e) In vitro packaging into lambda phage.

(f) Transfecting E. coli cells with lambda phage and selection.

(g) Carrying out small scale plasmid isolations on tetracyclineresistant colonies and digesting to check that foreign DNA had beencloned.

(h) Plating and screening for colonies which clear chitin.

(i) Characterizing cosmid clones conferring chitinase activity.

(a) Isolation of total S. marcescens DNA

Cells of Serratia marcescens QMB1466 were removed from culture storageand streaked on agar media to form single isolated pure colonies.

A single colony was inoculated into 5 mls of 1% bactotryptone, 0.5%yeast extract and 0.5% NaCl (hereinafter LB) liquid medium and grownovernight with shaking at 28° C. 1 ml aliquots were spun down in b 1.5ml Eppendorf tubes and resuspended in 0.3 ml 20 mM Tris, 10 mM EDTA (pH8.0). 0.1 ml of 5% SARKOSYL and 0.1 ml of 5 mg/ml pronase were added andthe cells were incubated at 37° C. for lysis to proceed for two hours.After this incubation, the solution was passed through a 19 gauge needleto shear the DNA slightly and thus to reduce the viscosity of thesolution. Next, 0.5 ml of phenol (pH adjusted to 8.0 with Tris) wasadded and the mixture shaken in the Eppendorf tube prior tocentrifugation. This step was repeated three times, with the supernatantfrom one centrifugation being re-extracted with fresh phenol. Then thesupernatant was extracted three times with 0.8 ml of a one-to-onemixture of phenol and chloroform/isoamyl alcohol (24:1) and once with0.8 ml of chloroform/isoamyl alcohol. The supernatant from this finalspin was brought to 0.3M sodium acetate and the DNA precipitated byaddition of 2.5 volumes of ethanol. After centrifugation to pellet theDNA precipitate, the DNA was redissolved in 0.1 ml of 10 mM Tris/1 mMEDTA (hereinafter TE). An aliquot was taken and diluted into 0.5 ml formeasurement of the optical density at 260 nm in order to find out theconcentration of nucleic acid. Typically this procedure permitted theisolation of 100-200 micrograms of DNA.

(b) Partial digestion of isolated DNA

The procedure adopted for establishment of appropriate DNA to enzymeratios for correct partial digestion was the widely used methoddescribed in Maniatis pp. 282-283. The objective was to establishconditions where the maximum fluorescence of the partial digestionproducts occurred in the size range 40-50 kb. 10 ug (microgram) of DNAwas incubated in 150 ul (microliter) of the restriction enzyme bufferspecified by the manufacturer (New England Biolabs) and dispensed in 15ul aliquots except for one tube which contained a 30 ul aliquot. 10units of EcoR1 were added to the 30 ul aliquot, the contents of the tubewere mixed and a 15 ul aliquot withdrawn, added to the next tube and thecontents mixed, and the procedure repeated down the series of tubes.After a one hour incubation at 37° C. the reaction was terminated with 3ul of 0.25M EDTA/50% glycerol/0.01% bromophenol blue, and the digestionproducts run on a 0.4% agarose gel which was stained with 0.5 ug/mlethidium bromide and examined by fluorescence in short wave uv light.The migration of the partial digestion products in the gel was comparedto size markers of known size. Once conditions were established forpartial digestion of chromosomal DNA to the appropriate degree, 200 ugof DNA was digested to this degree in an appropriately scaled-up volume.Partial digests giving weight average sizes at the maximum fluorescenceposition of 40 kb and 20 kb were mixed and fractionated on a glycerolgradient.

(c) Fractionation of partial digestion products by differentialsedimentation

The digestion was terminated by addition of enough 0.5M EDTA to bringthe final EDTA concentration to b 10 mM followed by incubation of thereaction at 65° C. for 10 minutes, and then was kept on ice until thegradient was loaded. An aliquot was checked for the degree of digestionbeing appropriate by running on a 0.3% agarose gel with DNA fragmentsize markers of appropriate size (e.g., digests of lambda DNA).

Linear gradients of 10-40% glycerol were prepared in 38 ml polyallomertubes. The 10% or 40% glycerol stock solutions were made up in 1M sodiumacetate, 5 mM EDTA. 0.5-1.5 ml aliquots of partial digests containing100-300 ug of partial digest were loaded on top of the gradients whichwere then spun at 25,000 rpm for 16 hours.

At the end of the centrifugation the tubes were punctured at the bottom,1 ml aliquots were dripped out and the DNA in them analyzed by agarosegel electrophoresis in 0.3% gels. Fractions containing DNA in the sizerange 22-32 kb were chosen for further work.

Fractions of this size range were pooled and dialyzed against 10 mM Tris1, mM EDTA (pH 8.0) for 24 hours with three buffer changes.

These fractions were then concentrated by isobutanol extraction(Maniatis, p. 463) to about 0.3 ml, brought to 0.3M sodium acetate andthe DNA was precipitated by addition of 2.5 volumes of ethanol. Theprecipitated DNA was redissolved in 10 ul of TE and the quantity of DNArecovered estimated by measuring the optical density at 260 nm of adilution of an aliquot of this DNA.

(d) Ligation of size-fractionated DNA to vector DNA

The vector used for cloning the Serratia DNA was pLAFRl. As described byA. Friedman et al., "Construction of a broad host range cosmid cloningvector and its use in the genetic analysis of Rhizobium meliloti", Gene,18, 289-96 (1982), pLAFRl has a single Eco RI site, a cos site fromlambda phage for in vitro packaging, and a tetracycline resistancemarker. The vector pLAFRl selects DNA inserts of about 22 to about 32 kbin length. The vector can be mobilized to other genera of bacteria whereit can replicate.

5 ug of pLAFRl DNA was digested to completion with EcoRl, and the DNAphenol extracted and ethanol precipitated. The precipitated DNA wasredissolved in 20 ul of TE.

Test ligations were carried out on both the pLAFRl DNA and thesize-fractionated DNA which was to be cloned to verify that the endswere ligatable.

In a typical ligation of pLAFRl DNA to size-fractionated Serratia DNA, a5-fold molar excess of Serratia DNA was adopted. A typical ligationcontained in 10 ul 0.4 ug of pLAFRl DNA and 3 ug of Serratia DNA. Thereaction was 66 mM Tris (pH 7.5), 10 mM MgCl₂, 1 mM ATP, 15 mMdithiothreitol, 0.05% BSA, 0.5 mM spermidine and 20 units/ul T4 DNAligase (New England Biolabs). The reaction was carried out overnight at15° C.

(e) In vitro packaging of ligation products into lambda phage particles

Packaging extracts were prepared as described in Maniatis p. 264-267.Freeze thaw lysate was frozen at -80° C. in 10 ul aliquots, and sonicextract was frozen away in 15 ul aliquots. One tube of each was thawedon ice and the freeze thaw was added to the sonic extract and mixedgently. Then 5 ul of the ligation was added to the mixture and aftergentle mixing, the packaging reaction was allowed to proceed at 25° C.for one hour. The reaction was diluted with 500 ul of b 10 mM MgCl₂ 10mM Tris (pH 7.5), 10 mM NaCl (hereinafter SM), and 500 ul chloroformwere added. The mixture was inverted five times in the capped Eppendorftube and spun for five minutes in an Eppendorf bench centrifuge.

(f) Transfection of E. coli cells with packaged cosmid clones

E. coli strain DHl (ATCC #33849) displays no detectable chitinaseactivity (see Table II). The strain was grown to saturation in LBcontaining 0.4% maltose. A 0.2 ml aliquot was withdrawn and mixed with0.1 ml of SM and 10 ul of the diluted packaging mix. After mixinggently, phage absorption was allowed to proceed for 20 minutes at 37° C.

The transfection was added to 1.7 ml of LB in a tube and the cellspermitted to grow out for 40 minutes at 37° C. In the first experiment20, 100, 500, and 1100 ul aliquots were plated on LB plates containing1.5% agar and 10 mg/1 tetracycline to investigate the colony formingunits derived from the packaging. The plates were incubated at 37° C.for 16-20 hours. In a typical experiment a 10 ul aliquot of packagingdilution would contain 1000 colony forming units

(g) Small scale plasmid preps to investigate the quality of the bank

Single tetracycline resistant colonies were picked from plates at theend of stage (f) above into 8 ml aliquots of LB containing 0.5 g/1uridine and incubated with aeration for 12-20 hours at 37° C. Cells werespun down and resuspended in 0.2 ml 50 mM glucose, 20 mM Tris, 10 mMEDTA (pH 8.0). The cells were then lysed in 0.4 ml 0.2M NaOH, 1% SDS.This was neutralized with 0.3 ml 3M potassium acetate which had beenbrought to pH 5.0 with acetic acid and incubated on ice for fiveminutes. After thorough mixing, the mixture was spun at 8000 rpm for tenminutes. The supernatant was precipitated with 0.6 volumes ofisopropanol and the DNA recovered by centrifugation in a bench topEppendorf centrifuge. The pelleted DNA was redissolved in 0.3 ml TE andextracted with equal volumes of phenol and chloroform. Aftercentrifugation the supernatant was brought to 0.3M sodium acetate andthe DNA precipitated with 2.5 volumes of ethanol. After centrifugationthe DNA was redissolved in 0.05 ml of TE and 5 ul aliquots were used forrestriction enzyme digestion followed by gel electrophoresis.

In a typical experiment eight independent clones were grown up and 50%of them contained inserts.

(h) Direct plating on chitin-containing medium for a screen forchitinase activity

Theoretically about 500 independent clones of a genome the size of E.coli should give a 99% chance of getting any particular sequence amongthe clones. It is desirable to independently isolate any clone withchitinase activity at least once. Five thousand colonies were plated outat about 250 colonies per plate on LB medium containing 2.0% colloidalchitin and 10 mg/1 tetracycline. This concentration of chitin had beenpreviously shown to clearly evidence the chitinase activity of S.marcescens QMB1466.

After about seven days at 32° C., certain colonies gave rise to clearzones around them. Altogether about twenty different colonies gave riseto convincing clear zones in their vicinity. See Table II and step ibelow. In Table II, DHl is the original E. coli (step f); DHl/pLAFRl isE. coli DHl containing the cosmid vector pLAFRl but without insert;DHl/C3 is E. coli DHl containing cosmid vector pLAFRl with one chitinasesize class insert; and DHl/Cl2 is E. coli DHl containing cosmid vectorpLAFRl with a second chitinase size class insert (this strain has beendeposited with the American Type Culture Collection in Rockville, Md. asATCC No. 67152).

(i) Characterization of cosmid clones conferring chitinase activity

Ten of the twenty colonies were inoculated into 8 ml of LB test mediumand plasmid DNA prepared as above. This DNA was analyzed for the DNAsequences in the plasmid by digestion with EcoRl. Each of the tenplasmid DNAs fell into one of two distinct size classes after EcoRldigestion. Seven out of the ten cosmid clones showed one large EcoRlfragment of about 25 kb, in addition to the vector band of 21.6 kb. Ofthese, one (C3) was chosen for further characterization (see Table IIand associated text). Three of the plasmids showed insert fragments of 3kb, 9.5 kb and 17 kb, in addition to the vector band of 21.6 kb. Ofthese, one (C12) was chosen for further characterization (see Table IIand associated text).

The phenotype of chitinase production was shown to be plasmid borne byreintroduction of the plasmid into E. coli bacteria by transformation ofthe bacteria with plasmid DNA. 1 ul out of the 50 ul of plasmid DNAprepared as in (h) was incubated with 0.1 ml of competent E. coli cellsprepared essentially by the method of M. Dagert and S. D. Ehrlich, Gene.6, 23-28 (1979). After a 20 minute incubation on ice and a two minuteheat shock at 37.C, bacteria were grown out in LB medium for one hourand plated on LB tetracycline chitin plates. Bacteria which acquiredtetracycline resistance all acquired the capacity to make chitinase.

The above experiments provide evidence that S. marcescens QMB1466contains two independent chitinase genes which have been isolated ondistinct cosmid clones. The means is therefore presented to expressthese genes either together or separately in a recipient organism.

2. Introduction of chitinase DNA into Pseudomonas species (a) Isolationof Pseudomonas species from the rhizosohere.

Pseudomonas fluorescens strain NZ130 and Pseudomonas putida strain MK280were isolated from radish roots, and soybean roots, respectively, byplating on King's Medium B from serial dilutions of root washings. Seein general T. Suslow, "Role of Root-Colonizing Bacteria in PlantGrowth," Phytopathogenic Prokaryates, Vol. 1, M. S. Mount and G. H. Lacyeds., 187-223 (1982) for details of fluorescent Pseudomonas isolationand characterization for colonizing ability and plant growth promotion.Strain NZ130 has been identified as P. fluorescens Biotype D(Pseudomonas chlororaphis in some taxonomies) and strain MK280 as P.putida. Their characteristics include the following:

                  TABLE I                                                         ______________________________________                                                            NZ130 MK280                                               ______________________________________                                        Fluorescent on King's Medium B                                                                      +       +                                               Fluorescent on King's Medium A                                                                      -       -                                               Pyocyanine Production -       -                                               Oxidase               +       +                                               Lecithinase           +       -                                               Gelatin Hydrolysis    +       -                                               Arginine Dihydrolase  +       +                                               Growth at 4° C.                                                                              +       ±                                            Growth at 37° C.                                                                             -       +                                               Growth at 41° C.                                                                             -       -                                               Green Phenazine Pigment                                                                             +       -                                               Motility              +       +                                               Inhibition of Erwinia sp.                                                                           +       +                                               Inhibition of Pythium sp.                                                                           +       -                                               Clones resistant to   +       +                                               rifampicin (100 ug/ml)                                                        ______________________________________                                    

Strain NZ130 has plant growth promoting characteristics on a number ofcrops including potato, radish, soybean, cotton, and sugar beet. StrainNZ130 also has biological control characteristics with respect toPythium sp., but no measurable chitinase activity. Root colonizationdata collected for NZl30, in general, reach average population densitiesof 5.5×10⁴ colony-forming units (cfu) per mg root tissue (dry weight) onradish, soybean and cotton.

Strain MK280 has been shown to increase the emergence of soybean and toeffectively colonize roots of soybean and sugar beets. Populationdensities, in general reach as high as 1.2×10⁶ cfu/mg root tissue.

(b) Mobilization of cosmid clones into Pseudomonas soecies

pLAFRl is a mobilizable cloning vector derived from pRK290 (Ditta). Itcan be mobilized into other genera of bacteria using a helper plasmidpRK2013 in a three-way mating process. Two Pseudomonas strains werechosen as recipients for these matings; these were NZ130r (NZ130,rifampicin resistant) and MK280r (MK280, rifampicin resistant). Thedonor (transferor) strain was E. coli DHl or HBl01 containing one of thetwo chitinase cosmid clones, and the helper strain was HBl01 containingpRK2013, all of which materials are commonly available.

Donor, recipient and helper strains were grown up to mid-log phase,without selection, in LB. 0.05 ml aliquots from each strain were addedto each other and the mixture put out as a 0.15 ml aliquot on an LBplate for 12-16 hours at room temperature. After the conjugation, a loopwas run through the cells. Cells from the loop were resuspended in 10 mMMgSO₄, and the mixture of cells plated at various dilutions on minimalsucrose tetracycline (10 mg/1) plates. This procedure selected againstE. coli, which cannot grow on minimal sucrose plates, and selects forPseudomonas cells which acquire the tetracycline resistance gene.Exconjugant cells were obtained and were tested for chitinase activityas above. See Table II. In Table II, TSO3l is NZl30r with cosmid vectorpLAFRl but without insert; TSO43 is NZl30r with cosmid vector pLAFRlwith one chitinase size class insert (corresponding to the insert ofDHl/Cl2 present in ATCC No. 67152); TSO35 is MK280r with cosmid vectorpLAFRl but without insert; and TSO44 is MK280r with cosmid vector pLAFRlwith one chitinase size class insert (corresponding to the insert ofDHl/C3). TSO44 has been deposited with the American Type CultureCollection in Rockville, Md. (ATCC No. 39637). Note that Table II alsolists data for two naturally occurring, chitinase producing strainswhich are not root colonizers: Serratia marcescens strain ATCC 990 and astrain of naturally occurring Arthrobacter sp.

                  TABLE II                                                        ______________________________________                                        Efficiency of Chitin Hydrolysis                                               by Chitinase Producing Bacteria*                                                       Growth Temperature                                                   STRAIN     21° C.                                                                         25° C.                                                                          28° C.                                                                       32° C.                                                                        37° C.                        ______________________________________                                        Serratia                                                                      marcescens                                                                    ATCC 990   1.28    1.51     2.17  2.22   1.41                                 Arthrobacter sp.                                                              TS037      1.0     1.06     1.07  2.0    1.0                                  E. coli                                                                       DH1        1.0     1.0      1.0   1.0    1.0                                  DH1/pLAFR1 1.0     1.0      1.0   1.0    1.0                                  DH1/C3     1.12    1.12     1.12  1.13   1.13                                 DH1/C12    1.12    1.12     1.12  1.32   1.82                                 P. fluorescens                                                                TS031      1.0     1.0      1.0   1.0    1.0                                  TS043      1.10    1.10     1.10  1.28   1.14                                 P. putida                                                                     TS035      1.0     1.0      1.0   1.0    1.0                                  TS044      1.12    1.06     1.06  1.14   1.15                                 ______________________________________                                    

3. Introduction of Chitinase DNA in Tobacco Under Control of a NopalineSynthase Promoter (a) Plant transformation

The coding region of the chitinase gene in the C12 chitinase size classinsert (described in Example 2 above) was determined by nucleotidesequence analysis of the DNA (this coding region referred to herein asthe chiA gene). See J. Jones et al., EMBO J., 5, 463-473 (1986)regarding sequence information. This nucleotide sequence information wasused in the creation of constructions which led to the expression of thebacterial chitinase gene in plant cells. Sequence information of use inpracticing the invention is set forth in the paragraphs below.

These constructions required the modification of nucleotide sequences atthe 5' end of the chitinase gene. This was carried out in several steps.An NdeI site (CATATG) was introduced at the N-terminalmethionine-encoding ATG of the chiA gene by oligonucleotidesite-directed mutagenesis; M. J. Zoller et al., Nucleic Acid Res., 10,6487-6500 (1985). Sequence information is given below.

    ______________________________________                                        chiA wild type sequence                                                                      GGAATCAGTT ATGCGC                                              chiA mutated sequence                                                                        GGAATCACAT ATGCGC (gives                                                      an NdeI site at the ATG)                                       ______________________________________                                    

The chiA gene was cut with NdeI and fused to a DNA fragment carrying theAorobacterium nopaline synthase (nos) promoter which had also beenmodified in a similar manner to yield an NdeI site at the ATG. Thenopaline synthase gene has been characterized; Depicker et al., J. Mol.Apol. Genet., 1, 561-573 (1982). Using these two constructions, the chiAgene was ligated directly to the nos promoter. Sequence information isshown below.

    ______________________________________                                        nos-chiA fusion TCTGCAT ATGCGCAAA                                             ______________________________________                                    

Modifications of the resulting fusion were made using site-directedmutagenesis in order to improve the translation signals. Sequenceinformation is shown below.

    ______________________________________                                        modified nos/chiA fusion #1                                                                    TCTGAAT ATGCGCAAA                                            modified nos-chiA fusion #2                                                                    TCTGAAT ATGGCCAAA                                            ______________________________________                                    

Each of these constructions was set up with a nos gene fragment carryingsignals for polyadenylation placed at an EcoRV site 25 bp past the TAA.These constructions were introduced into plant cells using Agrobacteriumby ligating them into binary vectors, P. van den Elzen et al., PlantMolecular Biology, 5, 149-154 (1985); and mobilizing the resultingconstructions into the Agrobacterium strain LBA4404, A. Hoekema et al.,Nature, 303, 179-180 (1983).

These strains were then used in cocultivation experiments with tobaccocells, and calli which grew on kanamycin were selected.

(b) Assays

Large numbers of independent calli (approximately 400) prepared asdescribed above were harvested approximately six weeks after initiatingthe selection regime and were analyzed for the expression of mRNAhomologous to the bacterial chitinase gene and for protein which wasantigenic to an antibody raised against the bacterial chitinase protein.This antibody was prepared using a protein sample in which the chitinaseband had been eluted from a preparative acrylamide gel and injected intorabbits to elicit antibodies to the protein in the rabbit serum. J.Jones et al., EMBO J., 5, 467-473 (1986).

The results of these experiments can be summarized as follows:

(i) Chimaeric nos-chitinase mRNA was detected in the transformed plantcells. About two-fold more mRNA was detected in modification #2 than inmodification #1 or in the original CATATG construction.

(ii) Bacterial chitinase protein was detected in the plant cells. Themodified nos-chiA fusion #2 gave rise to about four-fold more protein ona per total protein basis than modification #1 and eight-fold more thanthe original NdeI site fusion.

In short, these results showed the expression of chitinase protein inplant cells in detectable quantities.

4. Introduction of Chitinase DNA in Tobacco Under Control of aChlorophyll A/B Binding Protein Promoter (CAB Promoter) or a RibuloseBisphosphate Carboxylase Small Subunit Promoter (SSU Promoter) (a)Preparation of CAB constructs

The modified nos-chiA fusion #2 of Example 3 generated a Ballrestriction site (TGGCCA) immediately downstream of the ATG. This wasused to form a fusion with a chlorophyll a/b binding protein gene (CAB22L) octopine synthase fusion which also contained a Ba1I restrictionsite in a corresponding position relative to the ATG. See P. Dunsmuir etal., Nucleic Acid Res., 13, 2503-2518 (1985). This construction had theDNA sequence shown below.

    ______________________________________                                        CAB 22L/ ocs AAACC ATGGCCAGATCCCGGG                                           ______________________________________                                    

DNA carrying this construction was cut with Ba1I and ligated to BA1I cutnos/chitA modification #2 in such a way that the nos 3' polyadenylationsignals were retained 3' to the chiA gene. The resulting constructionhad a sequence around the ATG as shown below.

    ______________________________________                                        CAB-chiA fusion  AAA .sub.--C .sub.--C  .sub.--A .sub.--T .sub.--G                             .sub.--CCAAA                                                 ______________________________________                                    

(b) Preparation of SSU constructs

The resulting CAB-chiA fusion described above retained an NcoI (CCATGG)site at the ATG. This was used to fuse the chiA coding region to the 5'end of an expression cassette which used 5' and 3' sequences from a genehighly expressed in the petunia leaf, namely SSU gene #301; see C. Deanet al., EMBO J., 4, 3055-3061, (1985). This expression cassette(pAGS007) contains a coding sequence flanked by an NcoI site at the ATGand a BG1II (AGATCT), BamHI (GGATCC) or Bc1I (TGATCA) site close to and3' to the termination codon to be placed between SSU301 5' and 3'sequences. The expression cassette is deposited within E. coliJM83-AMB007 (ATCC 67125); see U.S. Ser. No. 883,604. Fusion to the SSUcassette creates a novel sequence in the vicinity of the chitinase ATG.Sequence information is given below.

    ______________________________________                                        SSU-chiA fusion  TAACC ATGGCCAAA                                              ______________________________________                                    

During the construction of the nos-chiA fusion the chiA coding regionfused to the nos 5' sequences was cloned as a BamHI (Klenow treated) toEcoRV (partial digestion) fragment into the SmaI site of a pUC vectorcontaining a TaqI fragment which encodes the nos 3' region. The EcoRVsite used was 25bp downstream of the chiA translation termination codon.This fusion therefore added the nos 3' region onto the 3' end of thechiA coding region, completing the nos-chiA fusion. The resultingplasmid had a BamHI site (from the pUC linker) at the junction of thechiA and nos 3' sequences. This BamHI site was used to fuse the 3'flanking sequences of the SSU expression cassette to the chiA codingsequence (a BamHI site can fuse to a BglII site).

(c) Plant transformation

The CAB and SSU constructions were cloned into the BamHI site of binaryvector pAGS135 which is very similar to pAGS112 described in P. van denElzen et al., Plant Molec. Bio., 5, 149-154 (1985). The difference isthat the unique XhoI site in pAGS112 has been removed by Klenow+dNTPtreatment of the linearized DNA and religation. The resultingconstructions were mobilized into Agrobacterium strain LBA4404. A.Hoekema et al., Nature, 303, 179-180 (1983).

The resultant Agrobacterium strains were cocultivated with protoplastsisolated from N. tabacum. P. van den Elzen et al., Plant Molec. Bio., 5,149-154 (1985). Transformed plant material was selected for its abilityto grow on 50 mg/l kanamycin, then regenerated.

The transgenic tobacco plants were transferred to the greenhouse oncethey had established a sufficient root system. They were assayed forexpression of chitinase RNA and chitinase protein three weeks after theyhad been in the greenhouse (previously established to be the time ofmaximal CAB and SSU RNA levels).

(d) Assay for chitinase protein

The level of chitinase protein was assayed by a Western blot analysis inwhich an antibody probe was used to detect chitinase protein on anitrocellulose filter carrying the size-fractionated polypeptides. Theamount of chitinase protein was evaluated using a standard dilutionseries of chitinase protein isolated from bacterial strains whichabundantly express the chitinase. These experiments showed that in themost abundantly expressing SSU-chitinase transformants, the bacterialchitinase protein accumulated to 0.1-0.2% of total leaf protein. Offifteen plants assayed, seven gave rise to greater than or equal to 0.1%of total protein as chitinase. For the CAB-chitinase transformant, onegave rise to a protein level of 0.1% chitinase, and on average theseconstructions gave rise to 2-4 fold less chitinase protein than wasfound in the SSU-chitinase transformants.

(e) Assay for chitinase mRNA

The levels of chitinase RNA were assayed by primer extension. In thisassay an oligonucleotide specific to the chitinase RNA was annealed tototal RNA and then extended in a reverse transcriptase reaction back tothe 5' end of the message. The amount of extended fragment then gave ameasure of the levels of chitinase RNA in the total RNA. The resultscorrelated broadly with the levels of chitinase protein observed in theindividual transformants. A comparison of the chitinase RNA levels fromplants transformed with noschiA, CAB-chiA and SSU-chiA showed that thehighest expressing SSU-chiA construct transformant gave rise to about15× more chitinase mRNA than the best CAB-chitinase constructs and atleast 200× more mRNA than the nos-chitinase constructs.

(f) Assay for chitinase biological activity

Biological activity of chitinase produced by transformants was assessedin a bioassay using, as a model disease, Tobacco Brown Spot caused byAlternaria alternata and Alternaria longipes. The pathogen infectsleaves causing discrete, readily quantified lesions and is of a class offungi, Fungi Imperfecti, having cell walls containing chitin.

Leaf disks, 9.0 cm in diameter, were cut from the center of tobaccoleaves taken from acropetal positions 4-10 as described by H. Spurr,Tobacco Science, 17, 145-148 (1973). Plants utilized were eithertransformed with a CAB: chiA fusion, 1771.2, or were transformed but didnot contain chiA, SBT2.9. The parental tobacco plant, Wis38, hadpreviously been tested for susceptibility to Brown Spot and ATCC strain26293 of A. longipes was most virulent. Conidial suspensions wereprepared as described (H. Spurr, Tobacco Science, 17, 145-148, 1973),and adjusted to approximately log₁₀ 5.0 conidia ml⁻¹ in a chamberhaemocytometer. Twelve drops (each of 10 ul) of conidial suspension wereplaced in a uniform pattern on the underside of each of seven leaf disksper treatment. Leaf disks were incubated in a Conviron Seed Germinatorwith settings at 21° C., 95% RH, and 8 hour low intensity fluorescentlight photoperiod.

After eight days, leaf disks were observed for comparative diseasedevelopment. Total necrotic lesions were counted and the lesion diametermeasured with a metric caliper. Diameter values given (Table III) areinclusive only of the brown necrotic tissue and not the chlorotic haloassociated with the whole infection. Leaf disks from identical leafpositions were most directly comparable as differences in susceptibilitywith leaf age were known.

Wis38 plants containing and expressing chiA had fewer total Brown Spotlesions, fewer lesions per disk, and necrotic areas which did developwere of generally lesser diameter than plants not transformed with chiA.

                  TABLE III                                                       ______________________________________                                               Total   Lesions/   a        b                                          Plant  Lesions disk       Diam./Total                                                                            Diam./Leaf                                 ______________________________________                                        1771.2 10      1.7        0.2      1.1                                        SBT2.9 23      3.3        0.6      1.5                                        ______________________________________                                    

In another series of readings, three other transformants (1781.1,1781.5, 1781.4) prepared as above, were compared with a control (Wis38).The results, shown in Table IV below, again show the positive effect ofchitinase, produced by the transformed plant, on disease resistance.Each of the three transformants showed, relative to the control,decreased infection ratio and decreased lesion sizes.

                  TABLE IV                                                        ______________________________________                                               Relative                      .sup.-- X diam./                                level of .sup.-- X infection                                                                      .sup.-- X diam./lesion/                                                                 lesion                                   Plant  chiA     ratio (%).sup.a                                                                          disk (mm).sup.b                                                                         (mm).sup.c                               ______________________________________                                        Wis38  0        82         6.4 ± 0.4                                                                            6.4 ± 0.20                            1781.1  1X      43         6.1 ± 0.5                                                                            6.2 ± 0.33                            1781.5  5X      62         5.6 ± 0.8                                                                            6.3 ± 0.38                            1781.4 20X      55         4.8 ± 0.4                                                                            4.6 ± 0.33                            ______________________________________                                         .sup.a Mean percent infection (infections/total inoculation points .times     100) per replication.                                                         .sup.b Mean infection lesion (necrotic area) diameter per leaf disk           replication.                                                                  .sup.c Mean infection diameter normalized per necrotic lesion            

What is claimed is:
 1. A method of inhibiting chitinous fungal pathogenscomprising introducing into a dicotyledonous plant a DNA sequenceencoding for chitinase activity to create a transformed plant underconditions whereby the transformed plant expresses chitinase in activeform.
 2. The method of claim 1 wherein said DNA sequence is isolatedfrom a bacterial source.
 3. The method of claim 1 wherein said DNAsequence is substantially homologous to chitinase-encoding DNA containedin ATCC #39637 or ATCC #67152.
 4. The method of claim 1 wherein said DNAsequence is fused to a plant promoter.
 5. The method of claim 1 whereinsaid DNA sequence is introduced using Agrobacterium.
 6. The method ofclaim 1 wherein the transformed plant expresses chitinase inbiologically active form as measurable by an assay using Tobacco BrownSpot.
 7. A transformed dicotyledonous plant resistant to chitinousfungal pathogens, said transformed plant containing a DNA sequenceencoding for chitinase activity and said DNA sequence having beenintroduced into the plant by transformation under conditions whereby thetransformed plant expresses chitinase in active form.
 8. The plant ofclaim 7 wherein said DNA sequence is isolated from a bacterial source.9. The plant of claim 7 wherein said DNA sequence is substantiallyhomologous to chitinase-encoding DNA contained in ATCC #39637 or ATCC#67152.
 10. The plant of claim 7 wherein said DNA sequence is fused to aplant promoter.
 11. The plant of claim 7 wherein said DNA sequence isintroduced using Agrobacterium.
 12. The plant of claim 7 wherein thetransformed plant expresses chitinase in biologically active form asmeasurable by an assay using Tobacco Brown Spot.